In vitro screening of anti-viral and virucidal effects against SARS-CoV-2 by Hypericum perforatum and Echinacea

Hypericum perforatum and Echinacea are reported to have antiviral activities against several viral infections. In this study, H. perforatum (St. John’s Wort) and Echinacea were tested in vitro using Vero E6 cells for their anti-viral effects against the newly identified Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) through its infectious cycle from 0 to 48 h post infection. The hypericin of H. perforatum and the different parts (roots, seeds, aerial) of two types of Echinacea species (Echinacea purpurea and Echinacea angustifolia) were tested for their anti-viral activities to measure the inhibition of viral load using quantitative real-time polymerase chain reaction (qRT-PCR) on cell culture assay. Interestingly, the H. perforatum-Echinacea mixture (1:1 ratio) of H. perforatum and Echinacea was tested as well on SARS-CoV-2 and showed crucial anti-viral activity competing H. perforatum then Echinacea effects as anti-viral treatment. Therefore, the results H. perforatum and Echinacea species, applied in this study showed significant anti-viral and virucidal effects in the following order of potency: H. perforatum, H. perforatum-Echinacea mixture, and Echinacea on SARS-CoV-2 infectious cycle. Additionally, molecular simulation analysis of the compounds with essential proteins (Mpro and RdRp) of the SARS-CoV-2 revealed the most potent bioactive compounds such as Echinacin, Echinacoside, Cyanin, Cyanidin 3-(6''-alonylglucoside, Quercetin-3-O-glucuronide, Proanthocyanidins, Rutin, Kaempferol-3-O-rutinoside, and Quercetin-3-O-xyloside. Thus, based on the outcome of this study, it is demanding the setup of clinical trial with specific therapeutic protocol.

The evaluation of herbal remedies and plant extracts that are shown to have an antiviral effect against other coronaviruses might provide an alternative approach to the development of COVID-19 treatments. Several studies have investigated the antiviral activities against other coronaviruses 14 with high efficacy and low cytotoxicity. For example, glycyrrhizin, an extract from licorice roots, was shown to completely block virus replication 15,16 .
H. perforatum (family Hypericaceae), or St. John's Wort (SJW) has been very well known for a long time as an effective medicinal plant for a range of communicable and non-communicable diseases such as depression, bacterial and viral infections, skin wound, and inflammation 17,18 . H. perforatum's metabolites extract from each plant part (roots, seeds and aerial) are different and are chemically defined to naphthodianthrones (hypericin), phloroglucinols (hyperforin), flavonoid glycosides (hyperoside), rutin, the flavonoids quercetin and myricetin 19,20 . As an anti-viral agent, H. perforatum activities were assessed in vitro and in vivo on infectious bronchitis virus (IBV), Hepatitis C, HIV, and Coronaviruses other than SARS-CoV-2 21,22 . Also, both hyperforin alone and other extracts of H. perforatum suppressed cytokine effects in β-cell lines and isolated rat and human pancreatic islets 20,23 . Furthermore, ethyl acetate extraction section of Hypericum (HPE) showed a significant reduction on relative virus titer of IBV in vitro and in vivo, reduction of mRNA expression rate of IL-6, TNF-α, and NF-kB 17 . Based on the potential of H. perforatum extract as well as its main polyphenol component hyperforin to counteract the pro-inflammatory effects of various cytokines, its use was reviewed and proposed to prevent cytokine storm in COVID-19 patients 24 .
Another medicinal plant, which was applied for many traditional and common remedies like curing cold and flu symptoms and boosting immune system, is Echinacea. Echinacea is known with nine species of several plants in the genus of Echinacea, however, only three of them were used as herbal complements: E. angustifolia, E. purpurea, and E. Pallida. Echinacea contains chemical compounds responsible for medicinal properties as: phenols including caffeic acid derivatives and echinacoside, polysaccharides, flavonoids, ketones, and lipophilic alkamides 25,26 . Previous in vitro and in vivo studies, showed that Echinacea had an effect on cytokine production 27,28 , increasing the expression of CD69 29 , an impact on natural killer cells 30 , as well as reducing illness severity. In vivo studies showed the anti-inflammatory therapeutic effect on human monocytic THP-1 cells 31 . Also, alkylamides and ketones of Echinacea extracts were reported for their anti-inflammatory effects [32][33][34][35] . A study in 2009 against H5N1 HPAIV strain showed that the extract of E. purpurea interferes with the viral entry into cells by blocking the receptor binding activity of the virus 36 . In another study, the in vitro virucidal and antiviral potential of Echinacea purpurea herb and roots ethanolic extract (Echinaforce®) was investigated against human coronaviruses including SARS-CoV-2 37 . The study reported the inactivation of MERS-CoV, SARS-CoV-1, and SARS-CoV-2 using the Echinacea purpurea extract.
In continuation to our previous studies to evaluate the antiviral performance of natural antiviral agents against some of the human coronaviruses 16,38,39 , we report in this study, the in vitro anti-viral and virucidal effects of H. perforatum (aerial parts) and Echinacea species (root, seed, aerial parts) against SARS-CoV-2. We also report a molecular simulation study for the reported bioactive compounds in the selected plants against the potent therapeutic targets, viz. main protein (M pro ) and RNA dependent polymerase (RdRp) enzymes of SARS-CoV-2.

Materials and methods
Cell cultures. Vero E6 cells (ATCC® CRL-1586™) and HEK 293 cells (ATCC® CRL-1573™) were incubated in a 75 cm 2 cell culture flask containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (SIGMA) at 37 °C in a 5% CO 2 atmosphere. For testing purposes, 96-well plates were seeded with Vero E6 cells at a density of 3 × 10 4 cells/well and incubated for 24 h at 37 °C with 5% CO 2 until a confluent monolayer was attained 40 . Viral stock. The SARS-CoV-2 isolate used in this study was isolated in the laboratory of BSL-3 from a well-characterized clinical specimen (SARS-CoV-2/human/SAU/85791C/2020, gene bank accession number: MT630432) described earlier 41 . Briefly, isolation was performed in a 75 cm 2 cell culture flask containing Vero E6 cells in Minimum Essential Media (Gibco, Thermo Fisher) 2 with 4% of fetal bovine serum and 1% glutamine. Cytopathic effect was monitored daily. In approximately 72 h, nearly complete cell lysis was observed yielding a TCID 50 of 3.16 × 10 6 infectious particles per mL. The viral supernatant was used for inoculation in subsequent experiments. All experiments involving live SARS-CoV-2 virus were performed in the biosafety level 3 facility of the Special infectious Agents Unit, King Abdulaziz University.
Hypericum perforatum and Echinacea extracts. H. perforatum and Echinacea products were purchased from gaia HERBS® (Brevard, North Carolina, USA) as gelatin capsules and as liquid; respectively, the products were sold as dietary and herbal supplements. According to the supplier, the whole plant profile is processed in the biocontainment area using water and ethanol to extract the plant's constituents to avoid applying non-ingestible solvents for extraction. Once extracted and filtered, the extract is then concentrated using low heat and low pressure to slowly remove the solvent and preserve the fragile plant constituents. Finally, HPLC analysis is carried to ensure the extract is concentrated to the correct activity levels.
Sample preparation. The plant materials in concentration of 100 mg/mL 42 were prepared in a BSL-2 laboratory under reduced light and dissolved in the minimum volume of dimethyl sulfoxide (DMSO, ≥ 99.5%, plant cell culture tested, SIGMA), diluted to the working concentration using culture medium and filtered into 0.22 µm filter 43 . All the extracts were filtered to remove any plant fibers. The mixture of H. perforatum and Echinacea was prepared by mixing each plant material at 100 mg/mL. . Cytotoxicity graphs were then generated by plotting percentage of cytotoxicity versus log10 the drug concentration using Graphpad prism 9 (Version 9.0.0) and the CC 50 was calculated using the nonlinear curve fitting with variable slope where the equation of the fit curve is: Y = 100/(1 + 10^((LogCC50-X)*HillSlope))) where Y is the % cytotoxicity and X is the concentration.
In vitro micro-inhibition assay. The plant extracts were evaluated as described previously 45 with some modifications. Briefly, 96-well plates were prepared as mentioned above with Vero E6 cells. Then, the cells were washed twice with PBS, and two-fold serial dilutions of plant materials (H. perforatum, Echinacea and the H. perforatum-Echinacea mixture) (0.316-5 µg/mL) in medium were challenged with a multiplicity of infection (MOI) of 1 of the SARS-CoV-2 isolate and incubated for three days at 37 °C and 5% CO 2 according to the procedures described in Section "Anti-viral activity assays". The results were quantified as previously described. The % inhibition was expressed relative to the virus control using dose-response curves. Graphs were then generated by plotting % inhibition versus log10 the drug concentration using Graphpad Prism 9 (Version 9.0.0) and the IC 50 was calculated using the nonlinear curve fitting with variable slope where the equation of the fit curve is represented by Eq. (1), where Y is the % inhibition and X is the concentration.
Anti-viral activity assays. The effect of the medicinal plants was tested in the BSL-3 biocontainment laboratory by the anti-viral assays (as represented in Fig. 1). The prepared 96-well plates of Vero E6 were treated with the herbal extracts in triplicate of two independent experiments: (1) Y = 100/(1 + 10 (LogIC50−X) * hillSlope )   46 , pooled together and used for qPCR. The percent inhibition of SARS-CoV-2 was evaluated relative to the positive virus control (SARS-CoV-2 on Vero E6 cells with no compounds added) 41 .  47 . The SARS-CoV-2 titers were expressed as PFU equivalents per mL (PEq/mL) using a standard curve generated by testing serial dilutions of the viral stock using qRT-PCR under the same testing conditions as the test samples. Each run included a positive viral template control and no-template negative control. Each sample was tested in duplicate, and the mean is reported as PEq/mL. Y = 100/(1 + 10^((LogIC50-X)*HillSlope)) where Y is the % inhibition and X is the con-centration 4.9.

Pre-treatment of virus prior to infection (virucidal activity
Statistical analysis. Data were analyzed with one-or two-way ANOVA with a Tukey's test for multiple comparisons. P < 0.05 and < 0.005 are considered statistically significant. All analyses were per-formed with GraphPad Prism, version 8. In silico study. Data  www.nature.com/scientificreports/ Molecular dynamics simulation. Selected re-docked complexes from each docking group were studied under 100 ns molecular dynamics simulation to understand the binding stability of phytochemicals in the active pocket of the selected viral proteins, i.e., SARS-CoV-2 M pro and SARS-CoV-2 RdRp, as reported earlier 57 . Briefly, the selected pose of docked complexes was pre-processed under default parameters using protein preparation tool in free academic Maestro-Desmond suite (Maestro-Desmond Interoperability Tools, Schrödinger, New York, NY, 2018) 58 . After that, the complexes were merged in orthorhombic water bath (10 × 10 × 10 Å) amended with TIP4P water solvent and the complete simulation system was neutralized by counter ions while ions were placed at a distance of 20 Å from the ligand using system building tool. Moreover, to mimick the in vitro physiochemical environment, 0.15 M salt was also amended in the simulation system by system building tool. Following, complete system was minimized using Desmond minimization tool 58 under default parameters and then subjected to 100 ns MD simulation at 300 K temperature and 1.01325 bar pressure using molecular dynamics simulation tool with default parameters. Each docked complex was subjected to MD simulation under similar parameters. Subsequently, the MD simulation trajectories were analyzed by simulation interaction diagram 59 tool in Maestro-Desmond suite 58 .

Results
Evaluation of cytotoxicity of Hypericum perforatum and Echinacea.  (Fig. S1). Further evaluation of the cytotoxicity of the plant extracts was performed on HEK293 cells as a human cell line. The cytotoxicity of the extracts was not concentration-dependent with the highest cytotoxicity for the Echinacea extract (Fig. S1).
Anti-viral efficacy of Hypericum perforatum and Echinacea. The antiviral effect of the medicinal plants was evaluated using qRT-PCR assay relative to the virus control as shown in Fig. S2 which shows the dose-response curve for the tested plants. The time response of the plants tested were evaluated through the following assays:

Post-treatment of virus-infected cells assay
Results from this assay showed that H. perforatum had the highest efficacy ( Fig. 2A) (Figs. 2 and 3) was evaluated in maximum non-toxic concentration (1.56, 6.25, and 6.25 µg/mL, respectively), and it showed that the inhibition of H. perforatum on SARS-CoV-2 was higher than H. perforatum-Echinacea mixture and Echinacea in the three anti-viral assays. Also, it displayed that Echinacea was a weaker inhibitor than the H. perforatum and the H. perforatum-Echinacea mixture but slightly strong as a virucidal effect up to 24 h. In addition, the effect of the plant materials on SARS-CoV-2 infection was evaluated by crystal violet staining of the viral CPE effect on the cells (Fig. 4). Figure 4 shows the effect of evaluating the antiviral activity of adding the extracts in the three different antiviral assays (Panels A, B and C) and followed up for 48 h post addition. PC is the positive virus control with no treatments added, while NC is the negative cell control with no virus and no treatment added.

Computational analysis.
To decipher the potential compounds, present in the respective extracts of selected plants, phytochemicals reported in each plant were collected from literature and studied for putative inhibitory mechanism with the aid of computational methods. This aid includes molecular docking, molecular contact formation, and molecular dynamics simulation.  These observed binding energies for the above compounds are much better than control ligand Remdesivir (− 7.6 kcal/mol), and some previously reported FDA approved drugs, which were repurposed against SARS-CoV-2 60,61 . This suggests that all the above reported compounds may be the potential inhibitors of both SARS-CoV-2 drug targets. The molecular interaction analysis results revealed the formation of good molecular contacts with catalytic residues and other substrate binding residues (Tables 1 and 2).  (Fig. 5)   www.nature.com/scientificreports/ observed interaction for each above discussed molecule is much better than the interaction reported in the crystal structure of SARS-CoV-2 M pro bound to potent broad-spectrum non-covalent inhibitor X77 because only three hydrogen bonds (with Gly 143 , His 163 , and Glu 166 ) had been observed in this determined structure of the complex and also in re-docked complex of SARS-CoV-2 M pro -X77 (Fig. S5) 50 . Likewise, interactions for the selected docked poses of SARS-CoV-2 RdRp with selected phytochemicals were also studied (Table 2). However, Interestingly, SARS-CoV-2 RdRp-Echinacoside showed 10 hydrogen bonds formation at Asp 452 , Thr 687 , Asp 760 , Asp 618 , Lys 551 , Arg 553 , Ala 554 , and Asp 623 residues while Asp 760 , Asp 452 , Arg 555 , Arg 553 , Lys 551 , and Asp 618 residues of SARS-CoV-2 RdRp formed 8 hydrogen bonds and three π-cation contacts (Arg 555 , and Lys 551 ) with Rutin from E. angustifolia (Fig. 6). In this study, Kaempferol-3-O-rutinoside from E. purpurea formed nine hydrogen bonds with Asp 452 , Lys 551 , Asp 623 , Lys 621 , Tyr 619 , Asp 618 , and Asp 760 residues of SARS-CoV-2 RdRp while Quercetin-3-O-xyloside from H. perforatum formed six hydrogen bonds and (with Asn 497 , Arg 569 , Gly 683 , Asn 543 ) and one pi-cation interaction (Lys 500 ) with SARS-CoV-2 RdRp (Fig. 6). Notably, a significant role of hydrogen bond formation between the receptor and ligand in the complex stability has been discussed and established in the field of drug discovery (please cite, Patil R, Das S, Stanley A, Yadav L, Sudhakar A, Varma AK. Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-designing 62,63 . Altogether, the calculated docking scores and intermolecular contact formation between RdRp and respective phytochemicals from E. angustifolia, E. purpurea, and H. perforatum suggested the screened compounds as potential inhibitors of SARS-CoV-2 RdRp by comparison to Remdesivir, potential inhibitors of SARS-CoV-2 RdRp, which was also reported with − 7.6 kcal/mol docking scores and interactions with similar active residues in SARS-CoV-2 RdRp 58,60,61 . Molecular dynamics simulation analysis. In the area of drug discovery, molecular dynamics simulation of the screened complexes from molecular docking is performed to understand the respective complex stability and intermolecular interactions with respect to time. In this study, selected docked complexes were studied for MD simulation interval via root mean square deviation (RMSD), root mean square fluctuation (RMSF), and proteinligand contacts mapping as function of 100 ns simulation interval.
RMSD analysis from MD trajectory of docked complex can be useful to understand the convergence of the complex with respect to time. Initially, protein (Cα) and protein fit ligands, i.e. selected phytochemical from three plants, were collected from the respective MD trajectories (Fig. 7). Notably, calculated RMSD values for alpha carbon (Cα) atoms of both SARS-CoV-2 M pro and SARS-CoV-2 RdRp showed < 3 Å acceptable deviations throughout the course of simulation. These observations suggested that the docked viral proteins have no significant structural changes as function of 100 ns interval (Fig. 7). Likewise, all the selected phytochemical as protein fit ligands with respective viral proteins showed substantial stability of < 5 Å, except SARS-CoV-2 M pro -Quercetin-3-O-glucuronide (< 6.5 Å), SARS-CoV-2 M pro -Proanthocyanidins (< 11.3 Å), and SARS-CoV-2 RdRp-Rutin (< 7.5 Å) exhibited higher deviations at the end of 100 ns simulation interval, suggested the substantial stability of the docked complexes (Fig. 7). Furthermore, these observations were also supported by acceptable fluctuations in protein RMSF (< 3 Å for SARS-CoV-2 M pro and < 6 Å SARS-CoV-2 RdRp) and protein fit ligand RMSF (< 3 Å in both proteins) values for the respective docked complexes, except higher < 6 Å deviations www.nature.com/scientificreports/ in SARS-CoV-2 RdRp in the region 300-325 residues in SARS-CoV-2 RdRp-Echinacoside and C-terminal in of the viral RdRp (Figs. S3-S4). Collectively, RMSD and RMSF analysis of the docked complexes supports the stability of the docked complexes in the active pocket of viral protease and RdRp during 100 ns MD simulation.
To further assess the stability of the docked complexes, protein-ligand interaction profiles, which include hydrogen bonding, hydrophobic interactions, ionic interactions, and water bridge formation, were extracted from the respective MD simulation trajectories (Fig. 8). Notably, the simulated complexes exhibited considerable molecular contact formation with the essential residues in the active pockets of the viral proteins during 100 ns simulation interval. Interestingly, the interacting residues were also noted in the initial docked poses of respective complexes (Tables 1,2). Of note, A significant contribution of hydrogen bond formation and water bridge assimilations were noted in SARS-CoV-2 M pro with selected phytochemicals while a substantial contribution of ionic interactions were also noted be-sides hydrogen and water bridge formation in SARS-CoV-2 RdRp docked with selected phytochemicals during simulation interval (Fig. 8). These protein-ligand contact maps, hence,

Discussion
COVID-19 pandemic is causing a global challenge to the world economic, social and healthcare systems. The pandemic is responsible for the death of over 5 million confirmed cases and 288 million infections worldwide 64 .
Although some vaccines are developed and are now being utilized under emergency use because of the pandemic, the efficacy of the vaccines is still debatable especially with the emergence of new variants of concern in the genomic structure [63][64][65][66] . Several non-specific treatment options were evaluated and entered clinical trials www.nature.com/scientificreports/ including the repurposing of known treatments against other diseases 3 . Therefore, improvement and investigation of an effective antiviral therapy is an urgent need for treating SARS-CoV-2 infection. In our study, the anti-SARS-CoV-2 effect of the medicinal plants H. perforatum, Echinacea and their combination was evaluated. The medicinal plants were purchased from a commercial source to ensure consistency of the composition and were tested either individually or combined together as a single treatment. Their mode of action was evaluated using three approaches namely post treatment of virus-infected cells, pre-treatment of cells prior viral infection and virucidal approaches. The cytotoxic effect of the tested medicinal plants was evaluated using MTT assay with results presented as percent of cytotoxicity relative to cell control (cells with no added tested medicinal plants). The CC 50 of H. perforatum and H. perforatum-Echinacea mixture was as follows 66.78 and 141.1 µg/mL; respectively while Echinacea was highly toxic.
The anti-viral activity of H. perforatum extract and hyperforin was previously reported to reduce the expression level of mRNA of IL-6 and TNF-α and to have a potent effect on the prevention of pro-inflammatory effect of numerous cytokines 20,23,63 . When exposed to light, hypericin showed different modes of anti-viral activities such as inhibition of budding of new virions 64 cross-linking of capsids preventing viral uncoating 65 , and inhibition of protein kinase activity required for replication of a number of viruses 66,67 . Moreover, it binds to phospholipids such as phosphatidylcholine of cell membranes, and it binds to retroviral particles, maybe by correlating with the membrane-derived lipid envelope 68 . Gibbons et al. noted that many hypericum species include biologically active acyl phloroglucinols 69,70 . Furthermore, polycyclic quinone of hypericin were reported to have an effective light-induced antiviral activity against many of enveloped viruses, including HIV-1 64,65,[71][72][73] . The molecular site of action of hypericin is increased > 100-fold in the presence of light 64,65,71-73 since it is a photosensitizing compound 74 . During illumination, singlet oxygen is efficiently produced with a quantum yield of 0.73 75 which is suggested to be the causative agent of hypericin's antiviral activity 64,65,72 or from complex mechanisms involving the superoxide anion and hypericin 76 . Other studies found 64,65 hypericin induces significant changes in the HIV capsid protein p24, in the presence of light, and may suppress reverse transcriptase activity.
Our results demonstrated that the H. perforatum, containing 0.9 mg/capsule of hypericins, can significantly reduce SARS-CoV-2 viral load compared to the positive control through the viral infectious cycle from 0 to 48 h (Table S13, Figs. 2, 3). This reduction is demonstrated in three mechanisms investigated in this study. Noticeably the results showed that the highest antiviral effect was through the virucidal mechanism (Fig. 3) compared to the other two mechanisms with the highest inhibition observed at 36 h after infection as shown in Fig. 2. While H. perforatum has the strongest inhibitory effect (35.77%) and reduction in viral load, up to 48 h, compared to Echinacea and the H. perforatum-Echinacea mixture that had 3.30 and 31.36%, up to 36 h, respectively. The lower inhibition of H. perforatum-Echinacea mixture than H. perforatum alone that would be expected as the active compounds in H. perforatum were diluted by the addition of Echinacea.
Hypericins were reported to be the active anti-viral compounds in H. perforatum extract against several viral infections both in vitro and in vivo including IBV 17 , bovine diarrhea virus (BVDV) 77 and hepatitis C virus (HCV) 47 with hypericin defined as the active ingredient. Other studies showed that H. perforatum extract had anti-viral effects against influenza A virus and HIV [78][79][80] . In addition to the anti-viral effect of H. perforatum extract, H. perforatum ethylacetate (HPE) extract showed a remarkable decrease in the concentration of IL-6 and TNF-α through the NF-κB in lung tissue of mice infected with an influenza A virus 80 and in the trachea and kidney of chickens infected with IBV, mainly from hypericin content of HPE 17 .
The observation that the highest inhibition was shown upon virus treatment followed by cells treatment before infection indicate the potential role that H. perforatum might play in virus-cell interaction (Figs. 2 and 3). H. perforatum also showed prolonged activity up to 48 h compared to H. perforatum-Echinacea mixture or Echinacea alone indicating its potential long-acting effect when used as antiviral against SARS-CoV-2.
Although Echinacea has been shown to have antiviral activity by other studies [81][82][83][84] , the addition of Echinacea to H. perforatum resulted in decreasing the antiviral activity. Studies have shown that Echinacea has a better effect as a cytokine regulator as shown by others 85,86 . Therefore an in vivo study is needed to evaluate the effect of Echinacea on cytokine regulation during COVID-19 infection together with the anti-viral effect found in this study of H. perforatum. The in vivo experiments should also investigate the anti-inflammatory effect of the mixture by measuring the mRNA expression levels of pro-inflammatory cytokines such as: IL-6, TNF-α, INF-β. This reduction in the inflammation is expected to empower the viral inhibition effect of the medicinal plants as reported in previous studies 17,20,23,31,34,37,46,48,63,72 .
In silico finding using molecular docking and MD simulation suggests that Echinacin and Echinacoside from E. angustifolia, Cyanin and Cyanidin 3-(6ʺ-malonylglucoside) from E. purpurea, and Quercetin-3-Oglucuronide and Proanthocyanidins from H. perforatum have good inhibitory potential against SARS-CoV-2 M pro via the inhibition of viral M pro proteins (Figs. 6, 8). Likewise, Echinacoside and Rutin from E. angustifolia, Kaempferol-3-O-rutinoside and Echinacoside from E. purpurea, and Rutin and Quercetin-3-O-xyloside from H. perforatum were identified to have good binding with SARS-CoV-2 RdRp (Figs. 6, 8). In hence, as observed in the in vitro assays (Fig. 1), identified phytochemicals from the selected plants were marked as potent inhibitors for main protease and RdRp, which are the essential proteins required in the initiation and replication of SARS-CoV-2 respectively.
Conclusively, Echinacoside and Rutin are considered as multi targeted compounds due to their good in silico inhibitory potential against both viral targets. Therefore, the identified phytomolecules can be considered for further in vitro and in vivo validation for designing new potential drugs against SARS-CoV-2. As the standard treatment of COVID-19 is constantly changing with the new developments of antiviral therapies, future studies should include a comparison of the standard treatment at the time with the results from this study. www.nature.com/scientificreports/

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.